Though, the formation of the vertebrate vasculature of any organ system is a complex process that is orchestrated by a constellation of growth factors and guidance cues (Jain et al., 2003), recent studies have dramatically increased our understanding of the signaling cascades that regulate angiogenesis. For example, it is increasingly clear that molecular programs, which direct trajectory of axons and the formation of the neural network, have important roles in generating the highly stereotypical pattern of the mature vascular network (Carmeliet et al., 2005; Urness et al., 2004; and Jones et al., 2007).
During the initial phase of vascular development in mammals, which is referred to as vasculogenesis, endothelial cells differentiate, migrate and coalesce to form the central axial vessels, the dorsal aortae and cardinal veins. The second phase, called angiogenesis, is characterized by the sprouting of new vessels from the nascent plexus to form a mature circulatory system. VEGF (or VPF) is critical for both of these first two phases: the differentiation and survival of endothelial cells during vasculogenesis as well as proliferation and permeability during angiogenesis. Following this angiogenic remodeling, the endothelium secretes platelet-derived growth factor (PDGF), which induces the recruitment and differentiation of vascular smooth muscle cells. Subsequently, the vascular smooth muscle cells secrete angiopoietins, which ensure proper interaction between endothelial and vascular smooth muscle cells. Finally, the vascular smooth muscle cells deposit matrix proteins, such as elastin, that inhibit vascular smooth muscle cell proliferation and differentiation, thereby stabilizing the mature vessel. Thus, to establish and maintain a mature vascular network, the endothelial and smooth muscle compartments of a vessel must interact via autocrine and paracrine signaling. The gaps between endothelial cells (cell junctions) forming the vascular endothelium are strictly regulated depending on the type and physiological state of the tissue. For example, in a mature vascular bed, endothelial cells do not behave independently of one another; rather, they form a monolayer that prevents the movement of protein, fluid and cells from the endothelial lumen into the surrounding tissue.
Even after development, the vascular system is continually exposed to events, conditions or pathogens that cause injury, ischemia, and inflammation, which typically result in the release of cytokines and angiogenic factors, such as vascular endothelial growth factor (VEGF). Initially, VEGF was described, purified and cloned as vascular permeability factor (VPF), based on its ability to induce blood vessels to leak. VEGF destabilizes endothelial cell-cell junctions, leading to endothelial permeability, stimulates endothelial proliferation and migration, and promotes vascular sprouting and edema. These functions serve to deconstruct a stable vascular network producing leaky new blood vessels. In many contexts, the release of cytokines and angiogenic factors in response to injury, ischemia and inflammation is desirable, in that such a response leads initiates a restorative or healing processes. However, excessive angiogenesis and vascular leak (e.g., endothelial hyperpermeability) underscore the pathologies of several diseases and pathologic conditions.
For example, in the developed world, pathologic angiogenesis and endothelial hyperpermeability in the retinal or choroidal vascular beds are the most common causes of catastrophic vision loss. New and dysfunctional blood vessels leak, bleed or stimulate fibrosis that in turn precipitates edema, hemorrhage, or retinal detachment compromising vision. The major diseases sharing this pathogenesis include proliferative diabetic retinopathy (DR), non-proliferative diabetic macular edema (DME), and age-related macular degeneration (AMD) (Dorrell et al., 2007; Afzal et al., 2007). Approximately 15 million Americans over the age of 65 suffer from AMD, and 10% of these patients will experience visual loss as a result of choroidal neovascularization. Further, more than 16 million Americans are diabetic, and over 400,000 new patients suffer from retinal edema or neovascularization. Given that the current number of 200 million diabetics worldwide is likely to double in the next 20 years, and that over 8% of such patients suffer from microvascular complications, the number of patients that will experience vision loss from diabetic eye disease is unfortunately set to increase rapidly. Though less prevalent than DR, DME and AMD, retinopathy of prematurity (ROP) and ischemic retinal vein occlusion (IRVO) are also associated with pathologic angiogenesis and endothelial hyperpermeability in the retinal or choroidal vascular beds and lack effective treatment.
In addition to diseases of the eye, pathologic angiogenesis is also associated with tumor formation and growth. Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplying nutrients and oxygen and removing waste products. With angiogenesis tumor growth proceeds, without it, it stops. Tumor angiogenesis actually starts with cancerous tumor cells releasing molecules that send signals to surrounding normal host tissue. This signaling activates certain genes in the host tissue that, in turn, make proteins to encourage growth of new blood vessels. Angiogenesis is regulated by both activator and inhibitor molecules. Under normal conditions, the inhibitors predominate, blocking growth. However, during tumor formation and growth, tumor cells release angiogenesis activators, causing such activators to increase in number/concentration. Such an increase in angiogenesis activators results in the growth and division of vascular endothelial cells and, ultimately, the formation of new blood vessels.
More than a dozen different proteins, as well as several smaller molecules, have been identified as “angiogenic.” Among these molecules, two proteins appear to be the most important for sustaining tumor growth: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are produced by many kinds of cancer cells and by certain types of normal cells. VEGF and bFGF are first synthesized inside tumor cells and then secreted into the surrounding tissue. When they encounter endothelial cells, they bind to specific proteins, called receptors, sitting on the outer surface of the cells. The binding of either VEGF or bFGF to its appropriate receptor activates a series of relay proteins that transmits a signal into the nucleus of the endothelial cells. The nuclear signal ultimately prompts a group of genes to make products needed for new endothelial cell growth. The activation of endothelial cells by VEGF or bFGF sets in motion a series of steps toward the creation of new blood vessels. First, the activated endothelial cells produce matrix metalloproteinases (MMPs), a special class of degradative enzymes. These enzymes are then released from the endothelial cells into the surrounding tissue. The MMPs break down the extracellular matrix—support material that fills the spaces between cells and is made of proteins and polysaccharides. Breakdown of this matrix permits the migration of endothelial cells. As they migrate into the surrounding tissues, activated endothelial cells begin to divide and organize into hollow tubes that evolve gradually into a mature network of blood vessels.
Additional diseases and disorders characterized by undesirable vascular permeability include, for example, edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome, pericardial effusion, pleural effusion, acute lung injury, inflammatory bowel disease, ischemia/reperfusion injury in stroke, myocardial infarction, and infectious and non-infectious diseases that result in a cytokine storm. Though a cytokine storm is the systemic expression of a healthy and vigorous immune system, it is an exaggerated immune response caused by rapidly proliferating and highly activated T-cells or natural killer (NK) cells and results in the release of more than 150 inflammatory mediators (cytokines, oxygen free radicals, and coagulation factors). Both pro-inflammatory cytokines (such as Tumor Necrosis Factor-alpha, InterLeukin-1, and InterLeukin-6) and anti-inflammatory cytokines (such as interleukin 10, and interleukin 1 receptor antagonist) are elevated in the serum, and it is the fierce and often lethal interplay of these cytokines is referred to as a “cytokine storm.”
Cytokine storms can occur in a number of infectious and non-infectious diseases including, for example, graft versus host disease (GVHD), adult respiratory distress syndrome (ARDS), sepsis, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS). In the absence of prompt intervention, a cytokine storm can result in permanent lung damage and, in many cases, death. Many patients will develop ARDS, which is characterized by pulmonary edema that is not associated with volume overload or depressed left ventricular function. The end stage symptoms of a disease precipitating the cytokine storm may include one or more of the following: hypotension; tachycardia; dyspnea; fever; ischemia or insufficient tissue perfusion; uncontrollable hemorrhage; severe metabolism dysregulation; and multisystem organ failure. Deaths from infections that precipitate a cytokine storm are often attributable to the symptoms resulting from the cytokine storm and are, therefore, not directly caused by the relevant pathogen. For example, deaths in severe influenza infections, such as by avian influenza or “bird flu,” are typically the result of ARDS, which results from a cytokine storm triggered by the viral infection.
Because of its involvement in angiogenesis and vascular permeability, much attention has been focused on vascular endothelial growth factor (VEGF). Products that that reduce VEGF mediated angiogenesis and vascular edema are now marketed and available to patients. For example, the anti-VEGF antibody Ranibizumab (Lucentis), an antibody fragment of Bevacizumab (Avastin), which is itself a VEGF antibody (Rosenfeld et al., 2006; Brown et al., 2006) is commercially available for the treatment of AMD. The development and success of this product has triggered enormous commercial interest in alternative strategies for the treatment of diseases and conditions associated with pathlogic angiogenesis or enthothelial hyperpermeability. Other approaches for inhibiting VEGF signaling include, for example, anti-VEGF aptamer, a soluble VEGF receptor ectodomain, receptor tyrosine kinase inhibitors, and siRNA against either VEGF or its receptors. With respect to AMD, such strategies have shown promise. However, there remains tremendous interest in a similar approaches for treating other conditions associated with pathologic angiogenesis and vascular leak. Moreover, as VEGF is only one of many angiogenic, permeability and inflammatory factors that contribute to angiogenesis and vascular permeability, there is continued value in identifying pathways and developing methods that affect VEGF functionality as well as the functionality of other angiogenic, permeability, or inflammatory factors.